Method of detecting the cystic fibrosis transmembrane conductance regulator (CFTR) at cell exterior

Abstract

The present invention provides a recombinant nucleic acid encoding a modified cystic fibrosis transmembrane conductance regulator (CFTR) protein. The nucleic acid is modified by the insertion of a first heterologous segment encoding a detectable epitope in a region encoding an extracytoplasmic loop, such as at least one of EL1 through EL6. E12 is currently preferred. Proteins encoded by such nucleic acids, vectors and cells containing such nucleic acids, and methods of use thereof are also described.

Description

RELATED APPLICATIONS[0001]The present application is a National Phase Application of International Application Serial No PCT / US2004 / 018475, filed May 20, 2004, which in turn claims the benefit of Provisional Application Ser. No. 60 / 472,267; Filed May 21, 2003, the disclosures of which are hereby incorporated herein by reference in their entirety.FIELD OF THE INVENTION[0002]The present invention provides a method of detection of the cystic fibrosis transmembrane conductance regulator (CFTR) at the exterior of the cell, a high throughput screen for agents that promote transport to the cell surface plasma membrane of Δ508 CFTR and other misprocessed disease-associated CFTR mutants. The method of specifically labeling only the cell surface CFTR pool also enables its endocytosis and turnover to be quantified and thereby to also screen for agents that stabilize this pool.BACKGROUND OF THE INVENTION[0003]The CFTR protein exposes only ˜3% of its mass on the exterior surface of the plasma me...

AI Technical Summary

Benefits of technology

"The present invention provides a modified cystic fibrosis transmembrane conductance regulator (CFTR) protein that can be used to screen compounds for their ability to promote the transport of the protein to the cell surface and treat cystic fibrosis. The modification involves the insertion of a detectable epitope in a region encoding an extracytoplasmic loop of the protein. The invention also includes a vector that can be used to express the modified protein in a cell and a method of treating cystic fibrosis by administering a compound identified through the screening method."

Problems solved by technology

The CFTR protein exposes only ˜3% of its mass on the exterior surface of the plasma membrane making its detection difficult without permeabilization of the membrane.

However, both this study, where polyclonal antisera were generated against a synthetic peptide corresponding to the sequence of the first EL, and other work at Transgene S.A. where monoclonal antibodies were generated against a similar peptide, were never confirmed or shown to be of practical use in the ensuing decade.

Efforts in several other laboratories to generate EL1 antibodies capable of detecting CFTR from the cell exterior were unsuccessful.

Although it is known that unglycosylated CFTR is transported to the cell surface where it has some chloride channel activity, it is also known that it is expressed poorly compared to the native glycosylated molecule and has a much shorter lifetime.

Therefore it is not useful to monitor conditions that would favor transport of the ΔF508 CFTR polypeptide to the cell surface.

However, no details of how this was done were provided and results were shown only for Xenopus oocytes and not in a cell system amenable to high throughput screening (Konstas et al., 2002).

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